Cryo-electron microscopy has turned out to be particularly suitable for analysis of biological structures. In this technology, a hydrous sample is cryofixed; i.e., it is cooled very rapidly, avoiding the formation of ice crystals. The objects to be examined, such as cells, enzymes, viruses or lipid layers, are thereby embedded in a thin vitrified ice layer. The great advantage of cryofixation is that the biological structures are maintained in their native state and can be examined in their physiological environment. Among other things, cryofixation allows a biological process to be arrested at any desired time and to be examined in this vitrified state in the cryo-electron microscope.
Regardless of the type of sample preparation, transmission electron microscopic imaging requires that the sample be sufficiently thin. Samples used for the transmission electron microscope typically have a thickness of 30-100 nm, preferably of 50-80 nm. When using other transmission electron microscopic methods (e.g., intermediate voltage transmission electron microscopy (IVEM)), the samples may be significantly thicker. Samples of defined thickness can be obtained by sectioning using an ultramicrotome. In the process, a cryofixed sample is cut into very thin slices (cryosections). Another preparation method is to deposit thin liquid films on electron microscopic supports. In this method, a thin liquid film is frozen very rapidly, avoiding the formation of ice crystals. To this end, an electron microscopic support (“grid”) is immersed in a sample-containing liquid or, alternatively, the sample liquid is pipetted onto the support, excess liquid is removed, for example, using a filter paper, and the liquid film remaining on the support is cryofixed by plunging it into a bath of liquid ethane, for example. Cryofixed samples can be examined directly in the frozen state in a cryo-electron microscope, since they are able to withstand the high vacuum present in the electron microscope.
Automated and semi-automated cryopreparation devices used for cryofixation are known in the art. International Patent Application WO 02/077612 A1 (see also EP 1 370 846 B1 and US 020040157284) discloses such a device, which allows cryopreparation to be performed in a substantially automated manner. This device is marketed under the trade name Vitrobot™. In this device, the sample support is fixed in a holding device. Excess sample liquid is removed, if necessary, using a filter paper (blotting). Then, the support is rapidly plunged into a cryogenic bath (ethane), causing the sample to vitrify. Another device is produced by the Gatan Company (www.gatan.at) under the trade name Cryoplunge™. This device is simpler in construction and not fully automated.
In the known devices, the cryogen used for cryofixing the sample is located in a cryopreparation chamber which is open at the top (e.g., the liquid nitrogen workstation of the Gatan Cryoplunge™ device). Cooling of the cryogen is accomplished using an additional cryogen, typically liquid nitrogen. Further, the cryopreparation chamber is cooled by a stream of cold gas produced by evaporation of the liquid nitrogen.
After the cooling process, the sample support carrying the vitrified sample is transferred in several steps from the ethane into a cooled specimen holder for an electron microscope. This transfer of the cryofixed sample is very critical because contact with moist air causes the immediate formation of a layer of ice crystals on the sample. According to the common procedure, the sample support carrying the frozen sample is removed from the ethane and initially transferred into a transfer box (e.g., a grid box). This step is carried out in the above-mentioned cryopreparation chamber filled with cold nitrogen gas. The transfer box is in turn introduced into a metal container, which is typically cooled with liquid nitrogen. This metal container is then transferred into a loading station for a cooled specimen holder for an electron microscope, the mounting of the sample support in the cooled specimen holder being performed in the loading station. Both the insertion of the sample support into the transfer box, and the transfer of the transfer box into the loading station for the cooled specimen holder for an electron microscope (EM), are critical procedures and involve a potential for contamination.